Preliminary work establishes that three natural products-the polyketide-derived a-pyrone myxopyronin, the Dolyketide-derived a-pyrone corallopyronin, and the polyketide-derived macrocylic lactone ripostatin-inhibit Dacterial RNA polymerase (RNAP) through interactions with the RNAP "switch region," a structural element that mediates conformational changes required for RNAP to bind and retain the DMAtemplate in transcription. The compounds do not inhibit eukaryotic RNAP I, RNAP II, or RNAP III. The compounds ootently inhibit Gram-positive and Gram-negative bacterial growth, exhibit no cross-resistance with the nhibitors of bacterial RNAP in current clinical use in therapy of bacterial infection (therifamycin antibacterial agents, rifampicin, rifapentine, and rifabutin), and exhibit no cross-resistance with other inhibitors of bacterial RNAP under evaluation for future clinical use in therapy of bacterial infection. The proposed work wilt use x-ray crystallography, ensemble and single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, molecular cloning, surrogate-host expression, structure-based screening, and de novo screening, to address four specific aims: Specific Aim 1: Determination of structures of complexes of RNAP with switch-region-target inhibitors Specific Aim 2: Determination of mechanisms of inhibition of RNAP by switch-region-target inhibitors Specific Aim 3: Cloning, characterization, and surrogate-host expression of biosynthetic gene clusters for switch-region-target inhibitors Specific Aim 4: Identification and characterization of novel switch-region-target inhibitors The results will enable development of new broad-spectrum antibacterial agents that will be effective against bacterial strains resistant to currently used antibacterial agents. As such, the results will have direct relevance to public health and to development of countermeasures against bacterial strains that could be used in biowarfare or bioterrorism. In addition, the results will contribute to understanding RNAP structure and function and will provide tools for analysis of RNAP structure and function.